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1.
Static light scattering of high amylopectin waxy maize starch gently dispersed in 90% dimethyl sulfoxide–water yielded a weight average molecular weight Mw and radius of gyration Rg of 560×106 g/mol and 342 nm, respectively. To obtain an independent hydrodynamic characterization of these solutions, we measured the sedimentation coefficient for the main component in an analytical ultracentrifuge. The value of s0, the infinite dilution sedimentation coefficient, was 199 S. The translational diffusion coefficient D0 in very dilute solutions was measured by dynamic light scattering at 90° and found to be 2.33×10−9 cm2/s. An effective hydrodynamic radius Rh was calculated from this diffusion constant using the Stokes–Einstein equation and found to be 348 nm. The structure-related parameter ρ=Rg/Rh was calculated to be 0.98. The weight average molecular weight calculated from the Svedberg equation using the values measured for s0 and D0 was 593×106 g/mol. This result is in reasonable agreement with the light scattering results. As light scattering results are subject to experimental errors due to the possibility of dust contamination, the presence of microgel or aggregates, and the questionable applicability of light scattering theory to interpret results for macromolecular sizes approaching the wave length of light used as a source for scattering, it is advisable to have corroborating hydrodynamic data when possible to further validate light scattering results in this very high molecular weight range.  相似文献   

2.
Determination of the oligomeric state or the subunit stoichiometry of integral membrane proteins in detergent solution is notoriously difficult, because the amount of detergent (and lipid) associated with the proteins is usually not known. Only two classical methods (sedimentation equilibrium centrifugation and static light scattering) can measure directly the absolute molecular mass of a protein present in a protein/detergent micelle, without any assumption on the amount of detergent bound, or the shape of the proteins. Here the theoretical background and practical aspects of static light scattering analysis of membrane proteins are reviewed using a number of examples from our lab to highlight potential pitfalls. A brief comparison with sedimentation equilibrium centrifugation is given and a detailed protocol of how we perform light scattering analyses is provided.  相似文献   

3.
Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 × 106 daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with Mw/Mn ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. The average value for β from the Mandelkern— Flory equation was 2.59 × l06, also agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky—Porod worm-like coil.  相似文献   

4.
Global conformation analysis of irradiated xyloglucans   总被引:2,自引:1,他引:1  
Xyloglucan isolated and purified from tamarind seed was subjected to various degrees of γ-irradiation treatments, from 10 to 70 kGy, monitored for radiation damage and then studied using a new combined hydrodynamic approach with regards to conformation and flexibility. Radiation products were analysed with regard to molecular weight (weight average) Mw from size exclusion chromatography coupled to multi-angle laser light scattering (SEC–MALLs), intrinsic viscosity [η] and sedimentation coefficient so20,w. Sedimentation coefficient distributions and elution profiles from SEC–MALLs confirmed the unimodal nature of the molecular weight distribution for each sample in solution. The chain flexibility was then investigated in terms of the persistence length, Lp of the equivalent worm-like chain model. The traditional Bushin–Bohdanecky (intrinsic viscosity) and Yamakawa–Fujii (sedimentation coefficient) relations were used separately then combined together by minimisation of a target function according to a recently published procedure [Ortega, A., & García de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475 [see also Ortega, A. Metodologías computacionales para propiedades en disolución de macromoléculas rígidas y flexibles. Ph.D. Dissertation, Universidad de Murcia, 2005]] and yielded an estimate for Lp in the range 4–9 nm using floated and fixed mass per unit length analysis protocols and “point” global analysis: irradiated xyloglucans behave as flexible structures in common with pressure/heat treated materials.  相似文献   

5.
Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using sedimentation equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from sedimentation equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from sedimentation equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.  相似文献   

6.
Statistical length of DNA from light scattering   总被引:7,自引:0,他引:7  
An analysis of hydrodynamic data of DNA by use of the wormlike coil model gave a statistical length of 1300 Å, in the absence of excluded volume corrections. The worm-like coil model and this hydrodynamically observed statistical length adequately describe the light scattering properties of DNA.  相似文献   

7.
The aim of this work is to examine the possible interaction and extent thereof of the polysaccharide galactomannan (GAL) with the cereal protein gliadin (GLI) and a peptic-tryptic degraded gliadin (PT-GLI) by analytical ultracentrifugation. The work is part of a series of investigations into the field of coeliac disease (gluten-induced enteropathy) as gliadins are known to be toxic for patients with this disease.

The molecular integrity of the GAL and GLI preparations was first checked by sedimentation velocity and sedimentation equilibrium. Sedimentation velocity showed single boundaries indicating homogeneity and low-speed sedimentation equilibrium gave plausible apparent weight average molar masses of 180,000 g/ mol for GAL and 20,000 g/mol for GLI. PT-GLI, GLI and GAL in phosphate buffer (pH 6.5) and the incubated mixtures (stirred for 3 h at 37 °C; PT-GLI: GAL = 3.53:1, wt.wt.; GLI:GAL = 0.23 and 0.55:1, wt.wt.) were then investigated by sedimentation velocity at a temperature of 20 °C. The plots of 1/s20 vs. c of GAL, PT-GLI-GAL and GLI-GAL mixtures after incubation show a significantly different shape suggesting the presence of interactions. According to the equation 1/s20 = 1/so20(1 + ksc), values for {so20, ks} of {(4.02 ± (490.9 ± 28.9) ml/g, {(5.92 ± 0.24) S, (1152 ± 44) ml/g} and {(5.38 ± 0.19) S, (1141 ± 38) ml/g} for GAL and PT-GLI-GAL and GLI-GAL mixtures, respectively, were obtained. The concentration of GAL ranged from 0.75–3.0 mg/ml for GAL alone and from 0.34–1.50 mg/ml in the incubated mixtures. This apparent indication for a weak non-covalent protein-polysaccharide interaction was further supported by UV absorption spectrometry and gel filtration.  相似文献   


8.
Formation of beta-amyloid plaques is a crucial feature of Alzheimer's disease. In the present work time resolved static light scattering was applied to investigate the size and shape of growing beta-amyloid aggregates preceding plaque formation. The beta-amyloid protein with 40 amino acid residues was used. Salt free buffer solutions and solutions with 0.15M NaCl at 37 degrees C served as the aggregation medium. The focus lay on the first 2h following initiation of the aggregation process which corresponds to the protofibril phase. Addition of the NaCl accelerated the aggregation process considerably. Scattering data from aggregation in saline solutions indicated formation of long fibers which suggest interpretation of data with the worm-like chain model. Two important results were revealed: (i) At the end of the time resolved recordings, the worm-like chain model provided a fully adequate picture for the growing aggregates. Chain stiffness is characterised in terms of the persistence length, which is close to 50 nm. The linear mass density of the growing fibers approached a value of two monomers per nm corresponding to single stranded fibers, which is in accordance with presently existing models for the aggregation of beta-amyloid. The fibers finally reached contour lengths of several thousand nanometers. (ii) The plateau values for the persistence length and linear mass density observed in the final regime are gradually approached from higher values. This observation is inconsistent with simple worm-like chains. Rather does it indicate existence of another species during the initial phase of the aggregation, in addition to monomers and fibers. Aside from further insight into fundamental aspects of beta-amyloid aggregation, time resolved static light scattering provides an appropriate tool for assay tests with drugs designed to interfere with the aggregation process.  相似文献   

9.
Solution properties of starch nanoparticles dispersed in DMSO and in water were studied using dynamic light scattering. The particle size distribution had two main peaks in both solvents at all scattering angles studied. They were at around 40 and 300 nm, ascribed to isolated starch nanoparticles and their aggregates, respectively. From the excess scattering intensity by the 40-nm particles, the molecular weight of the nanoparticle was estimated as 2.2–2.6×106 g/mol. When the concentration was increased, another peak appeared at around 1 μm. Raising the temperature from 25 to 65 °C did not change the distribution, indicating a purely entropic process in dynamic equilibrium of the aggregation. In DMSO, an oscillatory behavior was observed in the autocorrelation function at high temperatures.  相似文献   

10.
This article reviews some of the recent advances in analytical ultracentrifugation and how these advances have impacted--and can impact--on our understanding of the size, shape through conformation modelling, interactions and charge properties of polysaccharides in solution, particularly when used in combination with other solution techniques and also imaging techniques. Specifically we look at (1) polysaccharide polydispersity and simple shape analysis by sedimentation velocity, and in particular using new approaches such as SEDFIT analysis; (2) polysaccharide molecular-weight analysis by sedimentation equilibrium and MSTAR analysis and how this complements analysis of size exclusion chromatography coupled to multi-angle laser light scattering; (3) polysaccharide conformation analysis using traditional procedures such as the Wales-van Holde ratio, power law or 'scaling' relations, more specialised treatments for rigid cylindrical structures, semi-flexible chains and worm-like coils and complications through draining effects; (4) Analysis of polysaccharide interactions and in particular complex formation phenomena, focusing on interesting applications in the areas of mucoadhesion and sedimentation fingerprinting; and (5) the possibilities for macromolecular charge and charge screening measurement.  相似文献   

11.
Effects of Mg2+ ions on thermally induced conformational transitions in the synthetic poly(dA)·poly(dT) and poly(dA)·2poly(dT) were studied in the buffered solutions (pH 6.9), containing 0.1 or 1 M NaCl at polynucleotide concentration of 0.1–0.3 mM (in nucleic bases). The experiments consist of measurements of the UV absorption and intensity of conventional visible static light scattering. The diagram of conformational transitions in the poly(dA)–poly(dT)–Mg2+ system was constructed on a basis of experimental data obtained. Anomalously strong light scattering, like critical opalescence, has been revealed at 0.1 M NaCl and [Mg2+]≥20 mM in the melting range of both polynucleotides, which eventually disappeared after the completion of polymer strands separation. The effect presumably is caused by a fluctuation process of polymer strands complexing which arises at a certain concentration of Mg2+ ions.  相似文献   

12.
A detailed study of the interaction of chitin molecular species with the solvent system N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl) allowed the development of a new method for chitin fractionation by coacervate extraction. The controlled increase of the extracting power of the solvent was carried out using slight modification of the solvent composition. Partial extractions of molecular species were done between coacervation and complete dissolution limits using different mixtures of DMAc/LiCl of increasing extracting power. Fractions were characterized in DMAc/LiCl 5% (w/w) by viscometry and size exclusion chromatography with refractive index and multi-angle laser light scattering detectors. Fractions obtained by coacervate extraction range from 80,000 to 710,000 g mol−1 with polydispersity index between 1.28 and 1.44. The Mark–Houwink–Sakurada equation constants a and K for chitin in DMAc/LiCl 5% (w/w) were found to be 0.95 and 7.6×10−5 dl g−1, respectively.  相似文献   

13.
In 1% acetic acid, sedimentation velocity measurements and equilibrium ultracentrifuge experiments demonstrate that the Folch-Pi apoprotein is not monodisperse. The weight-average molecular weight calculated from ultracentrifuge experiments and combining sedimentation coefficient and viscosity measurements, ranged from 64000 to 80000. The intrinsic viscosity value suggests an asymetric shape for the apoprotein if a low value of hydration is considered. In dioxan/1% acetic acid (2:3, v/v) a smaller sedimentation coefficient was found, the intrinsic viscosity value remaining identical to that in 4% acetic acid. In pure 2-chloroethanol, light-scattering experiments led to a molecular weight of 165000 indicating that even in this solvent the protein is not monomeric. Intrinsic viscosity and light scattering measurements on the one hand, primary sequence on the other hand (six proline residues per monomer of Mr 23500) suggest that the molecule in 2-chloroethanol may consist of rod-like segments with flexible junctions.  相似文献   

14.
Studies on the molecular chain morphology of konjac glucomannan   总被引:2,自引:0,他引:2  
The chain geometry and parameters of konjac glucomannan were studied by using laser light scatter (LLS), gel permeation chromatography (GPC) and viscosimetry. The weight-average molecular weight (Mw), root-mean-square ratio of gyration (S21/2), second viral coefficient (A2) and polydispersity index (Mw/Mn) were 1.036×106, 105±0.9 nm, (−1.587±0.283)×10−3  mol ml g−2 and 1.015±0.003 respectively. Mark-Houwink equation was established as , and the molecular chain parameters were as follows: ML=982.82 nm−1, q=27.93 nm, d=0.74 nm, h=0.26 nm, L=1054.11 nm. To confirm the above results, konjac glucomannan was observed by using atomic force microscopy (AFM) and transmission electron microscope (TEM). The physical image showed directly that the konjac glucomannan molecule was an extending semi-flexible linear chain without branches, and than the molecular dimension also conformed to the parameters above. Therefore the image of molecular chain geometry confirmed the deduction drawn by Mark-Houwink equation and molecular chain parameters magnificently.  相似文献   

15.
Measurement of small-angle X-ray scattering from a sample of hyaluronic acid of high molecular weight in 0.05 m HNO3 gave persistence length plots which agreed in form with theory and led to apparent persistence lengths of from 4 to 6 nm. Similar measurements in 0.2 m NaCl gave plots which deviated somewhat in form from theoretical expectation, but which could be interpreted to give a persistence length of 4 nm in this solvent. Data for intrinsic viscosity [η] as a function of molecular weight were in reasonable agreement with the Yamakawa-Fujii treatment of [η] for the worm-like chain model for a persistence length of about 4 nm in both 0.5 m NaCl and 0.1 m HCl, perhaps slightly higher in the latter. The values of persistence length estimated from [η] depend somewhat on the choice of chain parameters and the method of correction of experimental data to unperturbed solvent conditions. Experimental data for the sedimentation coefficient, while less definitive, were consistent within experimental uncertainty with the same parameters of the worm-like chain model. These calculated results are in substantial agreement with the values derived from small-angle X-ray scattering. A fraction of hyaluronic acid of low molecular weight in 0.05 m HNO3 gave an estimated molecular weight of 2.7 × 104 and a radius of gyration of 8 nm, in reasonable agreement with expected values based on the worm-like chain model for a persistence length of about 4 nm.  相似文献   

16.
Dextran is currently being considered as a carrier biopolymer system for drug targeting. Di-iodotyrosine (DIT), and its radioactive derivative are useful markers for identifying the in-vivo route of these substrates. The performance of these substances in vivo is closely linked to their physico-chemical properties in solution. These properties have been investigated on a series of gel permeation chromatography fractionated DIT-dextrans using a combination of sedimentation, viscometric and laser-light scattering techniques. Weight average molecular weights, Mw, from sedimentation equilibrium and light scattering range from 0·0195 to 0·054 × 106. Double logarithmic representations of Mw versus sedimentation coefficient, so20.w, and intrinsic viscosity [η] suggest that these labelled dextrans have a more compact conformation in solution than do their unlabelled analogues — which behave as random coils. The implications of this for drug delivery are indicated.  相似文献   

17.
Semi-interpenetrating polymer network (IPN) microspheres of acrylamide grafted on dextran (AAm-g-Dex) and chitosan (CS) were prepared by emulsion-crosslinking method using glutaraldehyde (GA) as a crosslinker. The grafting efficiency was found to be 94%. Acyclovir, an antiviral drug with limited water solubility, was successfully encapsulated into IPN microspheres by varying the ratio of AAm-g-Dex and CS, % drug loading and amount of GA. Microspheres were characterized by FT-IR spectroscopy to assess the formation of IPN structure and to confirm the absence of chemical interactions between drug, polymer and crosslinking agent. Particle size was measured using laser light scattering technique. Microspheres with average particle sizes in the range of 265–388 μm were obtained. Differential scanning calorimetry (DSC) and X-ray diffraction (X-RD) studies were performed to understand the crystalline nature of drug after encapsulation into IPN microspheres. Acyclovir encapsulation of up to 79.6% was achieved as measured by UV spectroscopy. Both equilibrium and dynamic swelling studies were performed in 0.1 N HCl. Diffusion coefficients (D) and diffusional exponents (n) for water transport were determined using an empirical equation. In vitro release studies indicated the dependence of drug release rates on both the extent of crosslinking and amount of AAm-g-Dex used in preparing microspheres; the slow release was extended up to 12 h. The release rates were fitted to an empirical equation to compute the diffusional exponent (n), which indicated non-Fickian trend for the release of acyclovir.  相似文献   

18.
The Mg2+- and Ca2+-stimulated ATPase (bacterial coupling factor) has been investigated in solution with different independent techniques. The molecular weight of the five-subunit enzyme was found to be 345,000 +/- 5,000 by means of light scattering, 350,000 by sedimentation equilibrium experiments, and 358,000 by means of small-angle x-ray scattering. The radius of gyration was found to be 41.9 A, the volume 7.39 x 10(5) A3, and the surface to volume ratio 5.5 x 10(-2) A-1 from small-angle x-ray scattering measurements of the enzyme in solution. The degree of hydration was found to be 0.62 ml of H2O/g of ATPase. The translational diffusion coefficient was determined to be 3.47 x 10(-7) cm2 s-1 by means of inelastic light scattering. The distribution of the scattered intensity near the origin appears to be bimodal, suggesting that the ATPase molecule is composed of spherical parts bound together by a flexible polypeptide chain. The largest dimension of the ATPase in solution is 120.0 A, determined from the pair distribution function.  相似文献   

19.
A rapid method for establishing the constants in the Mark–Houwink–Sakurada equation, relating intrinsic viscosity and molecular weight (MW), of guar galactomannan is described. Following partial acid hydrolysis, the galactomannan was analyzed using high performance size exclusion chromatography employing viscosimetry and right angle light scattering detectors. In this way, a large number of samples of polysaccharides with a wide range of MW distributions were prepared, without need for isolation, and intrinsic viscosity and MW rapidly determined. The a and K values found for guar galactomannan were 0.72 and 5.13×10−4 ([η] in dl/g) respectively, in good agreement with previously published values.  相似文献   

20.
Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% -helix and 17% β-sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51 ± 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 ± 0.03 and 12.5 ± 0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V1 without C from Manduca sexta resulted in a V1–Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity.  相似文献   

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